The free radical scavenging capacity of the EO samples were measured using DPPH^o (2,2-diphenyl-1-picrylhydrazyl radical) as described by Mazidi et al. (2012). The IC₅₀ value is defined as the concentration of the antioxidant which is required to inhibit 50% of the DPPH° activity. Here, the IC₅₀ value was determined through graph plotting, by considering the percentage of the remaining DPPH° against the EO concentrations.

The ferric reducing power of EOs and that of the positive control (L-ascorbic acid) were determined here according to the method of Ardestani and Yazdanparast (2007). The reducing power was measured by reducing the Fe (III) to Fe (II). One mL of each EO solution (100–10000 mg·L⁻¹) was mixed with 2.5 mL of sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 10 g·L⁻¹ potassium ferricyanide (K₃Fe(CN)₆). The mixture was incubated at 50 °C for 20 min, whereupon 2.5 mL of 100 g·L⁻¹ trichloro acetic acid was added to the mixture and centrifuged for 10 min at 3000 g. The upper layer of solution (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl₃ (1 g·L⁻¹). The absorbance was measured by the spectrophotometer at 700 nm. Generally, a higher absorbance value indicates a higher reducing power. Results were expressed as mg ascorbic acid equivalents per gram of sample.

The cupric ion reducing power of the EOs and the positive control (L-ascorbic acid) were determined here in a test tube by mixing together 1 mL of CuCl₂ solution (10 mM), 1 mL neocuproinemethanolic solution (7.5 mM) and 1 mL ammonium acetate aqueous buffer solution (1 M). EO sample solutions (0.50 mL, 100–10000 mg·L⁻¹) and H₂O (0.60 mL) were added to the initial mixture so that the final volume reaches 4.10 mL. The tubes were stoppered and, after 30 min, the absorbance was recorded against the blank at 450 nm (Apak et al., 2008). Results are expressed as mg of ascorbic acid equivalents per gram of sample.

Free fatty acid content was determined according to the AOCS official method (Cd 3d-63) and was reported as a percentage of oleic acid (AOCS, 2000).

Fatty acid methyl esters were prepared here according to the method described by Golmakani et al. (2012). The composition and types of fatty acids in the VOO sample was analyzed using a GC system (SP-3420A, Beijing Beifen-Ruili Analytical Instrument, Beijing, China) which is a device equipped with a split/splitless injector, a flame ionization detector (FID) and a BPX70 capillary column (Bis–cyanopropylsiloxane-silphenylene, 120 m × 0.25 mm internal diameter; 0.25 μm film thickness, SGE Analytical Science, Melbourne, Australia). The temperatures of the column, injector and detector were set at 198 (isothermal), 250 and 300 °C, respectively. Nitrogen was used as the carrier gas. One μL of fatty acid methyl esters was then injected into the column with a split ratio of 1:10 accordingly. The fatty acids in the VOO were identified according to the retention times for standard fatty acids injected under the same operating conditions. The quantities of fatty acids were measured by calculating their relative peak areas.

Here, total phenols were isolated from a solution of oil in hexane by triple-extraction with water–methanol (60:40 v/v). The amounts of phenols were estimated using the Folin–Ciocalteu reagent at 725 nm. Results were expressed as mg of gallic acid per grams of VOO (Casal et al., 2010).

Chlorophyll and carotenoid contents were determined at 470 and 670 nm, respectively, according to the method described by Minguez-Mosquera et al. (1990).

The peroxide value (PV) was determined according to the AOCS official method (Cd 8–53) and was expressed as meq O₂·kg⁻¹ VOO. The p-Anisidine value (AV) was determined using the AOCS official method (Cd 8–53) and was expressed as mg·kg⁻¹ VOO (AOCS, 2000). The TOTOX value (total oxidation value; AV + 2 PV) is used as an empirical measure of the relevant precursors, the non-volatile carbonyls, present in the processed oils. The TOTOX value can also be used as a measure of any further oxidation products developed after storage (Frankel, 2012). The K₂₃₂ and K₂₆₈ extinction coefficients were determined according to the AOCS official method (ch 5–91) by measuring the absorbance of a pertinent solution (1% concentration) in isooctane at 232 and 268 nm, with 1 cm of pass length (AOCS, 2000).

Primary oxidation products, namely hydroperoxides, were determined by PV measurement. Changes in the PVs of VOO samples during storage at 70 °C are illustrated in Figure 1. The PV of the control increased gradually until the 35^th day, indicating the high resistance of VOO to oxidation due to its own (naturally occurring) antioxidants and low unsaturation level. However, the rate of hydroperoxide formation of the control increased sharply after reaching a PV of 38.44 meq O₂·kg⁻¹. This phase (after reaching a PV value of 38.44 meq O₂·kg⁻¹) indicated an accelerated degradation process. VOO supplemented with BHT, T. vulgaris EO, and B. persicum EO showed lower PVs in comparison with that of the control throughout the storage period. In the initial stages of oxidation, BHT and T. vulgaris EOs appear to be slightly more effective than B. persicum EO. However, the PVs of the samples containing BHT and T. vulgaris EO were significantly lower than that of B. persicum EO at the end of storage. This phenomenon may be due to the fact that natural antioxidants in the VOO are consumed at the initial stage of oxidation. Also, this result may be related to the fact that the different magnitudes of antioxidant activities observed among the various antioxidants are more evident at later stages of oxidation.

The PV of the sample containing α-tocopherol was slightly lower than that of the control during the entire period of the experiment. The IP_PV of the control, BHT, α-tocopherol, T. vulgaris EO, and B. persicum EO samples were 16.86, 27.08, 18.05, 25.13, and 22.49 days, respectively.

The anisidine test is designed to measure high molecular weights of saturated and unsaturated carbonyl compounds in triacylglycerols (Frankel, 2012). Changes in AVs of VOO samples during storage at 70 °C are illustrated in Figure 1. During the storage, the increasing trend observed for AV was very similar to that obtained for PV.

The control sample exhibited the highest AVs during the entire period of the experiment. Adding T. vulgaris and B. persicum EOs offered protection to the VOO, inhibiting the formation of undesirable flavors emanating from secondary lipid oxidation processes. Similar to the PV results, the effects of natural and synthetic antioxidants on delaying the formation of secondary oxidation products was clearly determined at the end of storage, and the order of inhibitory effects of natural and synthetic antioxidants was BHT > T. vulgaris, EO > B. persicum, EO > α-tocopherol.

The TOTOX value is an indicator of primary and secondary oxidation products. The results of the TOTOX values of the VOO samples during storage at 70 °C are presented in Figure 1. BHT, T. vulgaris EO, and B. persicum EO all reduced the formation of primary and secondary oxidation products in VOO by 78.51, 69.01, and 53.99%, respectively, when considered at the end of storage. This indicates good capacity of both EOs to inhibit the oxidative process. Thymol and carvacrol are the major components of T. vulgaris EO, while γ-terpinene is one of the major components of B. persicum EO. These components have been reported to exhibit antioxidant properties (Ruberto and Baratta, 2000). Thymol and carvacrol are primary antioxidants which either delay or prevent the initiation step by reacting with a lipid-free radical or prevent the propagation step by reacting with the peroxy or alkoxy radicals (Yanishlieva et al., 1999), thereby retarding VOO oxidation. It has been previously reported that thymol is a better antioxidant in lipids than carvacrol, due to the greater steric hindrance capability of its phenolic group (Yanishlieva et al., 1999). The antioxidant activity of γ-terpinene is attributed to the presence of methylene groups in monoterpene hydrocarbons which is strongly active and may compete with the activated methylene in C-11 of linoleic acid (Ruberto and Baratta, 2000).

α-Tocopherol showed no measurable effect in reducing the TOTOX value of VOO. α-tocopherol is reported to possess slight degrees of antioxidant activity and can even be pro-oxidative at times. These results depend on the chemical concentrations and the temperatures applied (Marinova and Yanishlieva, 1992; Schuler, 1990). The threshold value for α-tocopherol as a pro-oxidant in extra VOO oxidation was 60 to 70 ppm during storage at 37 and 75 °C (Deiana et al., 2002). Also, it has been reported that the pro-oxidant activity of α-tocopherol tends to decrease as the temperature increases, even at its high levels of concentration (Marinova and Yanishlieva, 1992).

The formation of conjugated dienes in fats or oils gives rise to an absorption peak at 232 nm in the ultraviolet region (Frankel, 2012). Changes in K₂₃₂ of the VOO samples during storage at 70 °C are presented in Figure 2. A significant difference (p<0.05) in K₂₃₂ was observed between the control and the samples containing T. vulgaris EO and B. persicum EO. This indicates the significant antioxidant effect of both EOs (p<0.05). The K₂₃₂ value of BHT, T. vulgaris EO, and B. persicum EO exhibited identical increasing trends in the first 28 days of storage. After that, the K₂₃₂ value of the sample which contained B. persicum EO increased faster and reached 6.21±0.09 at the end of storage, whereas K₂₃₂ of BHT and T. vulgaris EO reached 4.2±0.98 and 5.3±0.07, respectively. The α-tocopherol had no measurable effect on causing a decrease in the formation of the conjugated dienes of VOO during storage at 70 °C.

Changes in K₂₆₈ of VOO samples are due to the formation of conjugated trienes (Figure 2). In line with the K₂₃₂ results, the levels of conjugated trienes at the end of storage were of lowest value in samples containing BHT followed by T. vulgaris and B. persicum EO, while the highest levels were found in the control and the sample containing α-tocopherol.

The durations of time that were required to reach the upper legal limits of K₂₃₂ (IP_K232) and K₂₆₈ (IP_K268) during storage at 70 °C are presented in Table 4. As expected, there was a strong correlation between IP_PV and IP_K232 (R²=0.985; y=0.432x+3.232). It is also understood that a strong correlation exists between IP_PV and IP_K268 (R²=0.967; y=0.444x+4.277). Moreover, IP_K232 and IP_K268 correlated directly with each other (R²=0.987; y=1.028x-0.589). However, in all samples, IP_K232 and IP_K268 values were lower than that of IP_PV (with the average percentage of difference being 41.51 and 48.19%, respectively). Also, it was separately observed that K₂₃₂ and K₂₆₈ reached the upper legal limits of 2.6 and 0.25, respectively, almost concurrently. This indicates that the monitoring of VOO oxidation in terms of K₂₃₂ or K₂₆₈ at 70 °C will lead to the maintenance of stability in the VOO with regard to relatively similar quantities.

IP_PV, IP_K232, and IP_K268 were increased by the four antioxidants used in this study by approximately the same order as described before (BHT > T. vulgaris, EO > B. persicum EO > α-tocopherol). However, in all of the VOO samples under study, the IPE_PV value was higher than the values for the IPE_K232 and IPE_K268 measurements. This indicates that both natural and synthetic antioxidants are more effective in protecting MUFA than PUFA.

Chlorophyll compounds play an important role in the oxidative stability of VOO due to their antioxidant nature in the dark and their pro-oxidant activity in the presence of light (Criado et al., 2008). Carotenoids can act as primary antioxidants by trapping free radicals. They may also act as secondary antioxidants by quenching the singlet oxygen (Liebler, 1993). The chlorophyll contents of VOO samples during accelerated storage are presented in Figure 3. At the end of the storage period, all samples showed a substantial loss in chlorophyll content.

At the end of storage, the chlorophyll contents of VOO samples containing BHT, T. vulgaris EO and B. persicum EO ultimately decreased by 70.53%, 78.94% and 83.58%, respectively, whereas the chlorophyll content of the sample containing α-tocopherol and the control decreased by 89.68 and 90.91%, respectively. Carotenoid fractions decreased faster than the chlorophyll fraction during oxidation. It is commonly documented that the presence of oxygen and free radicals might accelerate the degradation rate of carotenoids. It is believed that the oxidation of carotenoids depends on the simultaneous oxidation of unsaturated fats (Criado et al., 2008). Thus, both oxygen and the presence of free radicals could explain the drastic decrease in carotenoid contents after a short period of storage. BHT, T. vulgaris EO, and B. persicum EO significantly retarded carotenoid degradation in the treated samples, compared with the control samples and also compared with those containing α-tocopherol. Nonetheless, at the end of storage, there were 28.37, 20.47%, and 13.49% carotenoids remaining in the samples containing BHT, T. vulgaris EO, and B. persicum EO, respectively, whereas the content of carotenoids was almost completely degraded in the control group and in the sample which contained α- tocopherol.